Central to the replication of HIV-1 is the precursor Gag polypeptide (p55) which is able to direct the assembly and release of virion-like particles even in the absence of all other virus encoded components. The p55 thus contains all of the necessary signals involved in directing its intracellular transport from its site of synthesis in the cytoplasm to the plasma membrane where virus formation and budding occurs. Accumulating evidence indicates that this intracellular assembly is a multi-step process controlled by several unique Gag polypeptide domains. We have used subcellular fractionation of cells expressing truncated Gag proteins to identify capsid and matrix polypeptide sequences possibly involved in p55 intracellular transport, membrane binding, and/or function. These include one domain which predicts an amphipathic alpha- helix and another which is phosphorylated by protein kinase C in response to cellular activation. The interaction of these domains with specific subceldular components (e.g., cytoskeleton, plasma membrane) as well as their response to general cellular activation signals will be investigated. The physiological significance of these specific domains in virion assembly and budding processes will be examined using mutant proviruses. Our overall objective is to characterize these newly identified domains with the goals of ascertaining their in vivo relevance to viral replication and their suitability as targets for chemotherapeutic intervention.